This invention relates to flow cytometry of the type in which a still picture of cells such as hemocytes in a flow of liquid is obtained and analyzed. The invention also pertains to an apparatus for practicing flow cytometry of this kind.
The conventional method of examining cells entails staining a smear of the cells on a glass slide and observing the cells under a microscope. Two problems are encountered in practicing this method. First, before the cells are observed under the microscope, it is required that a complicated procedure which includes smearing, fixing and staining the cells be followed to prepare the sample. Second, whereas cells such as hemocytes are present in a flowing liquid such as the blood when in a living body, with the conventional method these cells can only be observed in the fixed state and therefore do not present a true indication of the original cell morphology.
Recently, several attempts have been made to photograph the microscopical image of particles in a flow. For example, V. Kachel, et al., The Journal of Histochemistry and Cytochemistry, vol. 27, No. 1, p. 335 (1979) describe a method in which a still picture of particles is obtained by transmitting a suspension of the particles through an orifice, sensing the transmission of the particles through the orifice by a variation in electrical impedance at such time, triggering a flash lamp at the instant the particles are sensed and simultaneously photographing the particles. Further, the specifications of Japanese Patent Publication (KOKOKU) No. 57-500995 and Japanese Patent Application Laid-Open (KOKAI) No. 58-76740 teach a method of obtaining a still picture of particles in a liquid flow by passing a liquid specimen through a passageway, triggering a strobe at a fixed cycle in an imaging region and simultaneously photographing the particles with a CCD camera.
These attempts at photographing the microscopical image of particles in a flow involve a number of problems and shortcomings. The Kachel method requires the combining of resistive and optical detection principles and therefore necessitates a detector set-up which is structurally complex and difficult to adjust. Likewise, the method disclosed in Japanese Patent Publication No. 57-500995 relies upon a structurally complicated flow cell for forming the imaging region, and additional complications are encountered in producing the specimen flow. Furthermore, both methods use a flash lamp or strobe light known to emit an irradiating light pulse of comparatively large pulse width. In order to obtain a still picture, therefore, the flow velocity of the specimen carrying the particles of interest cannot be made very high. This places a limitation upon the processing capability of the system.
Neither of the aforementioned methods deals with image analysis of the internal cell structure and with the utilization of the information that might be obtained. Thus, these methods not only are incapable of making genuine cytometry possible but are not designed for such purpose.